A molecular imaging nano-probe for glioma boundary determination and a preparation method thereof

Abstract

The application discloses a kind of molecular imaging nano-probes for glioma boundary determination and preparation method thereof.The nano-probe comprises dendritic polymer, tumor targeting material and gadolinium connected on the surface of the dendritic polymer respectively;The tumor targeting material is the inhibitor of targeting acetyltransferase GCN5;The tumor targeting material is connected with the dendritic polymer by PEG;The gadolinium is connected with the dendritic polymer by chelating agent;The dendritic polymer is polyamide-amine type dendrimer or polylysine dendritic molecule.The nano-probe of the application can specifically target glioma, has good body safety, in vivo stability, provides a new imaging method for in vivo determination of glioma boundary, evaluation of curative effect and grading, brings a new way for precise diagnosis and treatment of glioma, and has great scientific significance and potential application value.

Description

Technical Field

[0001] This invention relates to the field of medical diagnostics, and in particular to a molecular imaging nanoprobe for determining the boundary of gliomas and its preparation method. Background Technology

[0002] Gliomas are the most common malignant tumors of the central nervous system. Due to their special location, in addition to their rapid growth and rich blood vessels, as the tumor itself progresses, it exhibits a biological behavior of highly invading and infiltrating the surrounding healthy brain tissue, making the boundary between the glioma and the surrounding brain tissue unclear.

[0003] Finding effective diagnostic and treatment methods must be based on a deep understanding of the pathophysiological and molecular mechanisms of glioma development and progression. Extensive clinical practice shows that early diagnosis and treatment of gliomas are mutually reinforcing; accurate early diagnosis is beneficial for long-term patient survival. Early detection and successful delineation of the glioma lesion facilitate accurate diagnosis and targeted treatment, thereby minimizing tumor tissue removal and damage to surrounding brain tissue. Therefore, effectively distinguishing the biological boundaries between glioma tissue and normal brain tissue, determining tumor grade, and accurately displaying the extent of glioma infiltration remain key issues in glioma diagnosis and treatment.

[0004] Currently, imaging examinations (such as CT and MRI) are the main means of clinically detecting gliomas. However, early-stage gliomas often lack obvious clinical signs, making accurate diagnosis difficult with imaging examinations, and even more difficult to clearly show tumor boundaries. This makes it difficult to remove glioma cells infiltrating the boundary between the tumor and normal brain tissue during surgery or radiotherapy. In recent years, molecular imaging technology has developed rapidly, and new molecular imaging biomarkers are constantly emerging. These biomarkers are usually genes or proteins highly associated with tumors. When good imaging techniques are available, the expression of these molecular markers can determine the tumor grade, effectively distinguish between the tumor and adjacent tissues, accurately define the tumor boundary, guide surgery and radiotherapy, and predict tumor prognosis. Regarding early diagnostic targets, a large amount of basic research has screened numerous mutated genes and protein targets, forming target libraries. Especially in the post-genomic and proteomics era, thousands of tumor targets have been discovered. Translational medicine research is expected to screen molecular imaging targets or multiple combinations of targets from the target library for early diagnosis. In terms of molecular probe construction, radiolabeling methods based on small molecules, macromolecules and nanoparticles have been established to obtain a series of intelligent responsive molecular probes, dual-targeting (fusion peptide) probes and multimodal probes.

[0005] Despite the rapid development of molecular imaging technology, progress in visualizing the biological boundaries of gliomas remains unsatisfactory. The main reason for this is that the presence of the blood-brain barrier makes it difficult for many molecular imaging contrast agents to cross this barrier and reach the glioma growth site, hindering their use in monitoring the biological boundaries of gliomas.

[0006] Therefore, developing molecular imaging probes that can effectively cross the blood-brain barrier in glioma patients and have glioma targeting capabilities remains an urgent priority for glioma treatment. Summary of the Invention

[0007] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide a molecular imaging nanoprobe for glioma boundary determination, its preparation method, and its uses. This invention discovers and tracks the key driver gene acetyltransferase GCN5, which is involved in the development and progression of gliomas, enabling early diagnosis, boundary delineation, and dynamic imaging monitoring of glioma evolution. By utilizing this novel glioma target to construct a nanoprobe-specific imaging platform, specific imaging of gliomas is achieved, improving the localization and boundary delineation of gliomas, as well as subsequent efficacy evaluation.

[0008] To achieve the above objectives, the present invention provides a molecular imaging nanoprobe for determining the boundary of gliomas, the nanoprobe comprising: a dendritic polymer, a tumor-targeting substance and gadolinium respectively attached to the surface of the dendritic polymer;

[0009] The tumor-targeting substance is an inhibitor that targets acetyltransferase GCN5;

[0010] The tumor-targeting substance is linked to the dendritic polymer via PEG;

[0011] The gadolinium is linked to the dendritic polymer via a chelating agent;

[0012] The dendritic polymer is a polyamide-amine type dendritic molecule or a polylysine dendritic molecule.

[0013] Optionally, the tumor-targeting substance is 9-(4-hydroxy-3-methoxyphenyl)-3,4,6,7,9,10-hexahydroacridine-1,8(2H,5H)-dione (DC-G16) and its derivatives, wherein the structural formula of DC-G16 and its derivatives includes the following:

[0014]

[0015] Optionally, the tumor-targeting substance is DC-G16-11. The structural formula of DC-G16-11 is:

[0016]

[0017] Optionally, one end of the tumor-targeting substance is connected to PEG, and the other end of the PEG is connected to maleimide.

[0018] Optionally, the dendritic polymer contains 20 to 80 amino groups.

[0019] Optionally, the dendritic polymer has a particle size of 5–30 nm. After loading tumor-targeting substances and gadolinium onto the surface of the dendritic polymer, the particle size remains almost unchanged, and the particle size of the dendritic polymer is approximately equal to the particle size of the nanoprobe.

[0020] Optionally, the molecular weight of the PEG is 1,000 to 40,000; more preferably, the molecular weight is 1,000 to 3,000; and even more preferably, the molecular weight is 2,000.

[0021] Optionally, the chelating agent is a metal ion chelating agent having a macrocyclic ligand. Preferably, the chelating agent is any one of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), Nota, and DTPA. More preferably, the chelating agent is linked to a maleimide.

[0022] Optionally, the gadolinium loading is 106–110 g / mg, based on the total mass of the nanoprobe.

[0023] The present invention also provides the use of the above-mentioned molecular imaging nanoprobe for glioma boundary determination in the preparation of products for tumor diagnosis or screening, wherein the tumor is highly expressed with acetyltransferase GCN5.

[0024] Optionally, the tumor is selected from breast cancer, pancreatic cancer, liver cancer, lung cancer, colon cancer, glioma, or leukemia; preferably, it is a glioma.

[0025] Optionally, the product is a contrast agent or a developer.

[0026] This invention also provides a method for preparing the above-mentioned molecular imaging nanoprobe for glioma boundary determination, specifically comprising the following steps:

[0027] S1, through a chelating agent, gadolinium is attached to the surface of the dendritic polymer to obtain an intermediate product;

[0028] S2, the intermediate product is mixed with the tumor-targeting substance in a solvent to obtain the nanoprobe.

[0029] Optionally, step S1 includes:

[0030] S1.1 causes gadolinium ions to chelate with the chelating agent to form a chelate product;

[0031] S1.2, the chelate product is mixed and reacted with the dendritic polymer, so that gadolinium is attached to the surface of the dendritic polymer as an intermediate product;

[0032] or,

[0033] S'1.1, the dendritic polymer is mixed and reacted with a chelating agent, so that the chelating agent is attached to the dendritic polymer;

[0034] S'1.2 then chelates with gadolinium ions, causing gadolinium to attach to the surface of the dendritic polymer as an intermediate product.

[0035] Optionally, it may include any of the following technical features:

[0036] The mass ratio of gadolinium to chelating agent is 2:(4-10);

[0037] The mass ratio of the chelating agent to the dendritic polymer is 1:(8-15);

[0038] In S1.1, the reaction temperature is 40–80℃;

[0039] In S1.1, the pH value is 4–7;

[0040] In S1.2, the reaction temperature is 10–40℃;

[0041] In S1.2, the pH value is 7–10;

[0042] In S'1.1, the reaction temperature is 10–40℃;

[0043] In S'1.1, the pH value is 7–10;

[0044] In S'1.2, the reaction temperature is 40–80℃;

[0045] In S'1.2, the pH value is 4 to 6.

[0046] In S2, the mass ratio of the intermediate product to the tumor-targeting substance is (15-30):1;

[0047] In S2, the reaction temperature is 10–40℃;

[0048] In S2, the pH value is 7-10.

[0049] Compared with the prior art, the present invention has the following beneficial effects:

[0050] 1) The molecular imaging nanoprobes of the present invention for determining the boundary of gliomas, in glioma cell lines, blood-brain barrier biomimetic chips, and mouse orthotopic xenograft models, demonstrate that they can visualize the malignant evolution and determine the biological boundary of gliomas on high-field MRI, visualize the evolution of gliomas in vivo, and accurately delineate their biological boundaries, which helps in imaging to grade gliomas and evaluate the efficacy of subsequent treatments.

[0051] 2) The nanoprobe obtained by this invention has good stability and high targeting in vivo, providing a new imaging method for determining the boundary of glioma in vivo, evaluating the efficacy and grading, and also bringing new ways to the precision diagnosis and treatment of glioma, which has great scientific significance and potential application value. Attached Figure Description

[0052] Figure 1 The diagram shows a schematic of the molecular imaging nanoprobe for glioma boundary determination according to the present invention.

[0053] Figure 2 The flowchart shown is a method for preparing the molecular imaging nanoprobe for glioma boundary determination according to the present invention.

[0054] Figure 3 The image shown is a transmission electron microscope (TEM) image of Den-Gd-DC-G16, a molecular imaging nanoprobe for glioma boundary determination obtained in Example 1 of the present invention.

[0055] Figure 4 The image shows the distribution of Gd in the Den-Gd-DC-G16 molecular imaging nanoprobe for glioma boundary determination obtained in Example 1 of the present invention. Each green dot represents the energy spectrum data of Gd, and the image shows gadolinium in the field of view separately.

[0056] Figure 5 The diagram shows the hydrated particle size distribution of the intermediate product Den-Gd obtained in Example 1 of the present invention.

[0057] Figure 6 The image shown is a hydrated particle size distribution map of the Den-Gd-DC-G16 molecular imaging nanoprobe for glioma boundary determination obtained in Example 1 of the present invention.

[0058] Figure 7 The diagram shows the zeta potentials of the dendritic polymer Den, the intermediate product Den-Gd, and the nanoprobe Den-Gd-DC-G16 in Example 1 of the present invention.

[0059] Figure 8 The diagram shows the relaxation rate curve of the nanoprobe Den-Gd-DC-G16 in Example 6 of the present invention.

[0060] Figure 9 The graph shows the survival rate of U251 cells at different time points after treatment with the nanoprobe Den-Gd-DC-G16 in Example 7 of this invention.

[0061] Figure 10 The image shown is a T1-weighted MR image at different time points after tumor-bearing nude mice were injected with the nanoprobe Den-Gd-DC-G16 in Example 8 of the present invention. Detailed Implementation

[0062] The technical solution of the present invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0063] This invention discovered acetyltransferase GCN5, a key driver gene in the development and progression of gliomas, which is highly expressed in glioma sites. Therefore, this invention identified a small molecule inhibitor that specifically binds to GCN5, namely DC-G16 and its derivatives. The functional targeting group is 9-(4-hydroxy-3-methoxyphenyl)-3,4,6,7,9,10-hexahydroacridine-1,8(2H,5H)-dione. The derivatives were further modified to improve the in vivo distribution efficiency of the probe and enhance its binding efficiency to GCN5.

[0064] The performance requirements for molecular imaging nanoprobes used for glioma boundary determination include at least: 1) the ability to cross the blood-brain barrier; 2) the ability to specifically bind to GCN5; and 3) biocompatibility. This invention designs a probe that can cross the blood-brain barrier, accumulate at the glioma site, and precisely delineate the glioma boundary using imaging.

[0065] This invention selects dendritic polymers as carriers based on the following criteria: 1) Good particle size controllability and uniformity, allowing the nanoprobe particle size to be controlled below 50 nm. Dendritic polymers carrying DC-G16 nanoprobes also possess some lipophilicity, meeting the requirements for crossing the blood-brain barrier. 2) Specific binding to GCN5 requires that the materials used to prepare the probes be easily modified with inhibitors that specifically bind to GCN5 (DC-G16 and its derivatives). Therefore, this invention selects polyamide-amine dendritic molecules or polylysine dendritic molecules as carriers, which are rich in amino groups, easily modified, and possess good biocompatibility.

[0066] like Figure 1The diagram shows the structure of the molecular imaging nanoprobe for glioma boundary determination according to the present invention. Den represents a dendritic polymer, and DC-G16 is an inhibitor targeting acetyltransferase GCN5. The nanoprobe comprises a dendritic polymer (Den), an inhibitor (DC-G16) attached to the surface of the dendritic polymer, and gadolinium (Gd) attached to the surface of the dendritic polymer; DC-G16 is connected to Den via PEG; and Gd is connected to Den via a chelating agent (e.g., DOTA).

[0067] like Figure 2 The diagram shows a flowchart of the preparation method of the molecular imaging nanoprobe for glioma boundary determination according to the present invention. Den, Mal-DOTA, and gadolinium trichloride react to obtain the intermediate product Den-Gd. The intermediate product Den-Gd reacts with DC-G16 in HEPES (as a buffer, or water or water-containing solution) to obtain the nanoprobe DEN-Gd-DC-G16 of the present invention. Figure 2 As shown, the specific steps include:

[0068] S1, gadolinium is attached to the surface of the dendritic polymer using a chelating agent to obtain an intermediate product; the mass ratio of gadolinium to the chelating agent is 2:(4-10). The mass ratio of gadolinium to the chelating agent can be 2:(4-6), 2:(5-8), or 2:(7-10). In a preferred embodiment, the mass ratio of gadolinium to the chelating agent is 2:5. The mass ratio of the chelating agent to the dendritic polymer is 1:(8-15). In some embodiments, the mass ratio of the chelating agent to the dendritic polymer can be 1:(8-10), 1:(9-12), 1:(11-13), or 1:(12-15). In a preferred embodiment, it is 1:10.

[0069] S2, the intermediate product is mixed with the tumor-targeting substance in a solvent to obtain the nanoprobe. The reaction temperature is 10-40°C. The mass ratio of the intermediate product to the tumor-targeting substance is (15-30):1. Preferably, the mass ratio of the intermediate product to the tumor-targeting substance is (15-25):1, or (20-30):1. In a preferred embodiment, it is 20:1. The reaction temperature can be 10-25°C, 20-35°C, or 30-40°C. In a preferred embodiment, it is 25°C. The pH value is 7-10. Preferably, the pH value can be 7-9, or 8-10. In a preferred embodiment, it is 8.4. The reaction time is 0.5-3 h. Preferably, the reaction time can be 0.5-1.5 h, 1-2 h, or 2-3 h. In a preferred embodiment, it is 1 h.

[0070] In some embodiments, step S1 includes:

[0071] S1.1, the gadolinium ions are chelated with a chelating agent to form a chelate product; the reaction temperature can be 40–80°C; preferably, the temperature can be 40–60°C, 50–70°C, or 60–80°C. In a preferred embodiment, the temperature is 60°C; the pH value is 4–7; further, the pH value is 4–6. Preferably, the pH value can be 4–5, 4.5–5.5, or 5–6. In a preferred embodiment, the pH value is 6. The reaction time is 10–60 min. Preferably, the time can be 10–30 min, 20–50 min, or 40–60 min. In a preferred embodiment, the time is 30 min.

[0072] S1.2, the chelated product reacts with the dendritic polymer, causing gadolinium to attach to the surface of the dendritic polymer as an intermediate product. The reaction temperature is 10–40°C. Preferably, the temperature can be 10–20°C, 15–30°C, or 25–40°C. In a preferred embodiment, it is 25°C. The pH value is 7–10. Preferably, the pH value can be 7–8, 7.5–9, or 8.5–10. In a preferred embodiment, the pH value is 8.4. The reaction time is 1–4 hours. Preferably, the time can be 1–3 hours or 2–4 hours. In a preferred embodiment, it is 2 hours.

[0073] In some embodiments, step S1 includes:

[0074] S'1.1, the dendritic polymer is mixed with a chelating agent and reacted, so that the chelating agent is attached to the dendritic polymer; the reaction temperature is 10–40°C. Preferably, the temperature can be 10–20°C, 15–30°C, or 25–40°C. In a preferred embodiment, it is 25°C. The pH value is 7–10. Preferably, the pH value can be 7–8, 7.5–9, or 8.5–10. In a preferred embodiment, the pH value is 8.4. The reaction time is 1–4 hours. Preferably, the time can be 1–3 hours or 2–4 hours. In a preferred embodiment, it is 2 hours.

[0075] S'1.2 is then chelated with gadolinium ions, causing gadolinium to attach to the surface of the dendritic polymer as an intermediate product. The reaction temperature is 40–80°C. Preferably, the temperature can be 40–60°C, 50–70°C, or 60–80°C. In a preferred embodiment, it is 60°C. The pH value is 4–6. Preferably, the pH value can be 4–5, 4.5–5.5, or 5–6. In a preferred embodiment, the pH value is 6. The reaction time is 10–60 min. Preferably, the time can be 10–30 min, 20–50 min, or 40–60 min. In a preferred embodiment, it is 30 min.

[0076] In this invention, DOTA refers to 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid (DOTA); HEPES refers to 4-hydroxyethylpiperazine ethanesulfonic acid.

[0077] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0078] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention.

[0079] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0080] In the following embodiments of this application: the dendritic polymer (Den) was purchased from Weihai Chenyuan Molecular New Materials Co., Ltd., and its molecular structure is as follows:

[0081]

[0082] Maleimide-DOTA, purchased from Hangzhou Xinqiao Biotechnology Co., Ltd., and labeled as Mal-DOTA.

[0083] In some embodiments, the inhibitor targeting acetyltransferase GCN5 is selected as DC-G16-11, which is modified with PEG at one end and maleimide at the other end of the PEG, denoted as Mal-PEG-DC-G16-11. Maleimide-polyethylene glycol-DC-G16-11 was synthesized by Shanghai Qiangyao Biotechnology Co., Ltd., and its structure is as follows:

[0084]

[0085] Example 1

[0086]

[0087] In this embodiment, the preparation of the nanoprobe includes the following steps:

[0088] Gadolinium trichloride was dissolved in 0.25M hydrochloric acid to form a hydrochloric acid solution with a final concentration of 50 mg / mL. 10 μL of the gadolinium trichloride hydrochloric acid solution was mixed with 1 mg of Mal-DOTA (molecular structure shown below), and the pH was adjusted to 6 with 0.25M sodium acetate solution. The temperature was raised to 60℃, and the chelation reaction was carried out for 30 min. Gadolinium was chelated by DOTA to obtain Mal-DOTA-Gd.

[0089]

[0090] Then, 20 mg / mL of 1 mL Den HEPES buffer solution was added to the chelated product Mal-DOTA-Gd. The pH of the HEPES buffer solution was 8.4. The mixture was stirred at room temperature (25°C) for 2 h. The Mal end of Mal-DOTA was bound to the amino group of Den via Michael addition reaction. After centrifugation, the intermediate product Den-Gd was obtained.

[0091] The 20 mg Den-Gd obtained above was mixed with 1 mg Mal-PEG-DC-G16-11 in HEPES buffer solution at pH 8.4 and reacted with stirring at room temperature (25 °C) for 1 h. DC-G16 was bound to the amino group of Den via a Mal-terminal Michael addition reaction. After centrifugation, the nanoprobe was prepared and labeled Den-Gd-DC-G16. Transmission electron microscopy was performed to observe the nanoparticles, and the particle size and zeta potential were measured.

[0092] Particle size determination: The sample was purified using a 0.45 μm pore size filter membrane and diluted to 100 g / mL with 1×PBS. Simultaneously, the equipment was calibrated using a 2.0 mg / mL bovine serum albumin standard solution. Particle size distribution was determined using a dynamic light scattering spectrometer. For determining the surface charge of the nanoprobes, the nanoprobe solution was filtered through a 0.45 μm filter and diluted to a 10 mM NaCl solution.

[0093] Zeta potential measurement: The surface potentials of Den, Den-Gd, and Den-Gd-DC-G16 in Example 1 were measured using a Malvern Zetasizer.

[0094] Figure 3 The images shown are transmission electron microscope (TEM) images of the nanoprobe DEN-Gd-DC-G16 in this embodiment. A is a TEM image of the nanoprobe Den-Gd-DC-G16 at 100 nm; B is a TEM image of the nanoprobe Den-Gd-DC-G16 at 50 nm. Figure 3 As can be seen from the above, the Den-Gd-DC-G16 nanoprobe obtained by this invention has a uniform morphology and is approximately 20 nm in size.

[0095] Figure 4 This is the EDS (Electromagnetic Spectrum Mapping) of the Den-Gd-DC-G16 nanoprobe in this embodiment. From... Figure 4 It can be seen that Gd was successfully chelated on the surface of the nanoprobe obtained by this invention.

[0096] Figure 5 This is a hydrated particle size distribution diagram of the intermediate product Den-Gd in this embodiment. From... Figure 5 It can be seen that the particle size distribution of the intermediate product Den-Gd is around 20 nm.

[0097] Figure 6 This is a hydration particle size distribution diagram of the nanoprobe Den-Gd-DC-G16 in this embodiment. From... Figure 6 It can be seen that the particle size distribution of the nanoprobe Den-Gd-DC-G16 is around 20 nm.

[0098] Figure 7 This is a zeta potential diagram of the dendritic polymer Den, the intermediate product Den-Gd, and the nanoprobe Den-Gd-DC-G16 in this embodiment. Figure 7 It is known that the surface potential of the dendritic polymer Den is 31 mV; after chelating Gd with the chelating agent Mal-DOTA, the surface potential of the intermediate product Den-Gd is 8.6 mV; after further connection with PEG-DC-G16, the surface potential of the nanoprobe Den-Gd-DC-G16 is -25.3 mV. The surface potential of the probe has changed significantly, indicating that Den and Gd of the present invention have been successfully connected with PEG-DC-G16.

[0099] Example 2

[0100] In this embodiment, the preparation of the nanoprobe includes the following steps:

[0101] 20 mg Den and 1 mg Mal-DOTA chelating agent were mixed in HEPES buffer solution at pH 8.4, stirred at room temperature (25°C) for 2 h, centrifuged, and the solid was collected to obtain Den-DOTA.

[0102] Mix 10 μL of 50 mg / mL gadolinium trichloride hydrochloric acid solution with 20 mg Den-DOTA, adjust the pH to 6 with 0.25 M sodium acetate solution, heat to 60 °C, react for 30 min, centrifuge to obtain the intermediate product Den-Gd.

[0103] 20 mg Den-Gd and 1 mg Mal-PEG-DC-G16-11 were mixed in HEPES buffer solution at pH 8.4, stirred at room temperature (25 °C) for 1 h, and centrifuged to prepare the nanoprobe Den-Gd-DC-G16.

[0104] Example 3

[0105] In this embodiment, the preparation of the nanoprobe includes the following steps:

[0106] Gadolinium trichloride was dissolved in 0.25M hydrochloric acid to form a hydrochloric acid solution with a final concentration of 50 mg / mL gadolinium trichloride. 10 μL of this gadolinium trichloride hydrochloric acid solution was then chelated with 0.8 mg of maleimide-DOTA chelating agent (Mal-DOTA). The pH was adjusted to 4 with 0.25M sodium acetate solution, and the mixture was heated to 40°C and reacted for 50 min to obtain Mal-DOTA-Gd. 20 mg / mL of 1 mL Den-10 HEPES buffer solution was added to the Mal-DOTA-Gd solution. The mixture was stirred at room temperature for 1 h, and then centrifuged to obtain the intermediate product Den-Gd.

[0107] The 20 mg Den-Gd obtained above was mixed with 1 mg Mal-PEG-DC-G16-11 in HEPES buffer solution at pH=10, stirred at 10℃ for 2 h, and centrifuged to prepare the nanoprobe Den-Gd-DC-G16.

[0108] Example 4

[0109] In this embodiment, the preparation of the nanoprobe includes the following steps:

[0110] Gadolinium trichloride was dissolved in 0.25M hydrochloric acid to form a hydrochloric acid solution with a final concentration of 50 mg / mL. 10 μL of this solution was then chelated with 2 mg of maleimide-DOTA (Mal-DOTA) chelating agent. The pH was adjusted to 7 with 0.25M sodium acetate solution, and the mixture was heated to 80°C and reacted for 20 min. Then, 20 mg / mL of 1 mL Den HEPES buffer solution (pH 7) was added, and the mixture was stirred at room temperature for 3 h. The mixture was then centrifuged to obtain the intermediate product Den-Gd.

[0111] The 20 mg Den-Gd obtained above was mixed with 1 mg Mal-PEG-DC-G16-11 in HEPES buffer solution at pH=10, stirred at 40℃ for 0.5 h, and centrifuged to prepare the nanoprobe Den-Gd-DC-G16.

[0112] Determination of Gd loading in nanoprobes

[0113] The method for determining the Gd loading in the nanoprobe obtained in Example 1 is as follows:

[0114] Dissolve 10 mg of the nanoprobe in hydrochloric acid, centrifuge, take the supernatant and dilute quantitatively to 10 mL, and determine the mass of Gd in the solution using an atomic absorption spectrophotometer.

[0115] The final gadolinium labeling rate was determined to be 108 ± 2 μg of Gd loading per unit mg of nanoprobe.

[0116] Relaxation rate determination of nanoprobes

[0117] The relaxation rate of the nanoprobe obtained in Example 1 was determined, including the following:

[0118] The nanoprobe Den-Gd-DC-G16 prepared in Example 1 was dispersed in 25 μL of water to prepare Den-Gd-DC-G16 aqueous solutions with final Gd concentrations of 0.5, 1.0, and 1.5 mM, with water as a control group. The solutions were then placed in relaxation tubes, and the T1 time of the Den-Gd-DC-G16 aqueous solutions at different concentrations was measured using a Bruker MQ60 relaxation analyzer.

[0119] Using 1 / T1 time as the ordinate, Gd was determined by atomic absorption spectrophotometry. 3+ Using concentration as the x-axis, a linear fit was performed to obtain the following result: Figure 8 The relaxation rate curve is shown. From... Figure 8 We know that y = 10.65x + 0.14(R) 2 =0.997), lateral relaxation rate r1 = 10.65mM -1 S -1 This demonstrates that the nanoprobe possesses magnetic properties and can be used for magnetic resonance imaging.

[0120] In vitro toxicity study of nanoprobes on cells

[0121] The nanoprobes prepared in Example 1 were subjected to in vitro cytotoxicity experiments, including the following:

[0122] Experimental group: U251 cells were seeded into 96-well plates at a density of 10,000 cells per well and incubated at 37°C for 24 h to allow cell attachment. Then, 1 mM of Den-Gd-DC-G16 (from Example 1) was added, and the cells were co-cultured at 37°C for 12 h, 24 h, and 48 h. The supernatant was then aspirated, and 100 μL of CCK-8 medium was added. The average absorbance of each well was measured at 425 nm using a microplate reader.

[0123] Control group: The Den-Gd-DC-G16 nanoprobe in the above experimental group was replaced with the same volume of double-distilled water.

[0124] The cytotoxicity of the nanoprobe Den-Gd-DC-G16 to cells was evaluated based on the ratio of the average absorbance of the experimental group to the control group.

[0125] Figure 9This is a graph showing the survival rate of U251 cells at different time points after treatment with the nanoprobe Den-Gd-DC-G16 in this embodiment. Figure 9 It was found that treating U251 cells with the 1mM nanoprobe Den-Gd-DC-G16 for 48 hours had almost no effect on cell viability and was therefore safe for human use.

[0126] Nanoprobes in vivo magnetic resonance imaging studies

[0127] The nanoprobes prepared in Example 1 were subjected to in vivo magnetic resonance imaging studies, including the following:

[0128] 1×10 7 U251 cells were suspended in PBS solution and subcutaneously injected into the right hind limb of 4-6 week old female mice to establish a U251 tumor xenograft model. Mice were fed until the tumor volume reached 100-120 mm². 3 Tumor-bearing mice were obtained.

[0129] The nanoprobe Den-Gd-DC-G16 obtained in Example 1 was dissolved in physiological saline to a probe concentration of 10 mg / ml. This solution was then injected into tumor-bearing mice via the tail vein, with each mouse receiving 300 μL of the probe at the above concentration (i.e., 3 mg of probe per mouse). T1-weighted magnetic resonance imaging (TMRI) was performed at 0.5 h, 3 h, and 6 h to observe the T1-weighted imaging effect at the tumor site.

[0130] Figure 10 T1-weighted MR images at different time points in this embodiment show the results of injecting the Den-Gd-DC-G16 nanoprobe solution into tumor-bearing nude mice. The baseline represents the state before injection. Figure 10 It can be seen that before injection, the boundary between the tumor tissue and the surrounding muscle tissue is not obvious; after injection, the tumor site becomes brighter, the MR signal is significantly enhanced, the magnetic resonance signal in the tumor area increases uniformly, and it is clearly distinguished from the surrounding tissue. Moreover, the signal becomes stronger over time.

[0131] MR images demonstrate that the nanoprobe of this invention can significantly improve the contrast effect of tumor sites, indicating that the nanoprobe of this invention has excellent targeting ability and can significantly enhance the magnetic resonance imaging effect.

[0132] The molecular imaging nanoprobes for glioma boundary determination provided by this invention can be used to prepare products for tumor diagnosis or screening, wherein the tumor highly expresses acetyltransferase GCN5. In some embodiments, the product is a contrast agent or imaging agent.

[0133] The tumor is selected from breast cancer, pancreatic cancer, liver cancer, lung cancer, colon cancer, glioma, or leukemia; preferably, it is a glioma.

[0134] In summary, this invention utilizes dendritic polymers (Dendrimer, Den) combined with the small molecule inhibitor DC-G16 of GCN5 and gadolinium (Gd) to synthesize a glioma-targeting nanomolecular probe, Den-Gd-DC-G16. Using this nanoprobe, magnetic resonance imaging (MRI) can be used to locate and precisely delineate the boundaries of gliomas in vivo, aiding in glioma grading and assessing the efficacy of subsequent treatments.

[0135] Although the present invention has been described in detail through the preferred embodiments above, it should be understood that the above description should not be considered as a limitation of the present invention. Various modifications and substitutions to the present invention will be apparent to those skilled in the art after reading the above description. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. A molecular imaging nanoprobe for determining the boundary of gliomas, characterized in that, The nanoprobe comprises: a dendritic polymer, a tumor-targeting substance and gadolinium respectively attached to the surface of the dendritic polymer; The tumor-targeting substance is DC-G16-11, an inhibitor targeting acetyltransferase GCN5; one end of the tumor-targeting substance is connected to PEG, and the other end of the PEG is connected to maleimide; The tumor-targeting substance is linked to the dendritic polymer via PEG; The gadolinium is linked to the dendritic polymer via a chelating agent; the chelating agent is linked to maleimide. The dendritic polymer is a polyamide-amine type dendritic molecule; based on the total mass of the nanoprobe, the gadolinium loading is 106-110 μg / mg; The dendritic polymer contains 20 to 80 amino groups. The chelating agent is DOTA.

2. The molecular imaging nanoprobe for glioma boundary determination as described in claim 1, characterized in that, Includes at least one of the following technical features: The dendritic polymer has a particle size of 5–30 nm; The molecular weight of the PEG is 1000-40000.

3. The use of a molecular imaging nanoprobe for glioma boundary determination according to claim 1 or 2 in the preparation of products for tumor diagnosis or screening, characterized in that, The tumor showed high expression of acetyltransferase GCN5.

4. The use as described in claim 3, characterized in that, The tumors mentioned include any one of glioma, breast cancer, pancreatic cancer, liver cancer, lung cancer, colon cancer, or leukemia.

5. The use as described in claim 3, characterized in that, The product in question is a contrast agent or imaging agent.

6. A method for preparing a molecular imaging nanoprobe for glioma boundary determination according to claim 1 or 2, characterized in that, The steps include the following: S1, through a chelating agent, gadolinium is attached to the surface of the dendritic polymer to obtain an intermediate product; S2, the intermediate product is mixed with the tumor-targeting substance in a solvent to obtain the nanoprobe.

7. The method for preparing the molecular imaging nanoprobe for glioma boundary determination as described in claim 6, characterized in that, Step S1 includes: S1.1 causes gadolinium ions to chelate with the chelating agent to form a chelate product; S1.2, the chelate product is mixed and reacted with the dendritic polymer, so that gadolinium is attached to the surface of the dendritic polymer as an intermediate product; or, S'1.1, the dendritic polymer is mixed and reacted with a chelating agent, so that the chelating agent is attached to the dendritic polymer; S'1.2 then chelates with gadolinium ions, causing gadolinium to attach to the surface of the dendritic polymer as an intermediate product.

8. The method for preparing the molecular imaging nanoprobe for glioma boundary determination as described in claim 7, characterized in that, Includes any one of the following technical features: The mass ratio of gadolinium to chelating agent is 2:(4-10); The mass ratio of the chelating agent to the dendritic polymer is 1:(8-15). In S1.1, the reaction temperature is 40–80℃; In S1.1, the pH value is 4–7; In S1.2, the reaction temperature is 10–40℃; In S1.2, the pH value is 7–10; In S'1.1, the reaction temperature is 10–40℃; In S'1.1, the pH value is 7–10; In S'1.2, the reaction temperature is 40–80℃; In S'1.2, the pH value is 4–6; In S2, the mass ratio of the intermediate product to the tumor-targeting substance is (15-30):1; In S2, the reaction temperature is 10–40℃; In S2, the pH value is 7-10.

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